Neutral protease I

Instructions
1. Solution preparation
1) Dissolve an appropriate amount of the freeze-dried powder of this product in DPBS buffer salt solution (free of calcium and magnesium ions) to prepare a stock solution of 10 mg/mL, and sterilize it by filtration with a 0.22 μM filter membrane.
2) When in use, dilute the above stock solution to the working solution concentration with DPBS. The commonly used working concentration for cell separation is 0.6-2.4 U/mL.
Note: It is not recommended to use working concentrations higher than 2.4 U/mL.
2. Dissociation of the organization
1) Use a sterile knife or scissors to cut the tissue into 3-4 mm tissue blocks.
2) Wash the tissue blocks with sterile PBS;
3) Add Dispase II solution (with a working concentration of 0.6-2.4 U/mL) to the tissue block and ensure that the tissue block is fully immersed in the Dispase solution.
4) Incubate at 37°C, stirring slowly during the incubation process until all tissue blocks are completely dissociated.
Note: Generally, for tissues that are difficult to dissociate, the separation goal can be achieved within 1 hour, but longer incubation periods (such as several hours) will not significantly affect cell activity.
5) If necessary, the above-mentioned digestion products can be filtered through a sterile stainless steel mesh screen to separate the single cells from the remaining tissue blocks. Or, after the large tissue has settled, gently pour out the upper layer of cells. If necessary, replace it with fresh dispase solution to further dissociate the remaining tissue.
6) Centrifuge the precipitated cells and discard the enzyme solution;
7) Resuspend the cell precipitate in the medium and culture the cells under normal conditions.
3. Cell passage
1) Immerse the cells in Dispase solution (preheated at 37°C) and incubate at 37°C for 5 minutes;
2) Aspirate the above solution and continue to incubate at 37°C for 10 minutes;
3) Observe the cell isolation under a microscope. If necessary, further incubate for 15 minutes.
4) Suspend the cells using the cell culture medium, gently rotate to make the cells settle, and then wash the cells with the culture medium.
5) Resuspend the cells in fresh cell culture medium and spread them out in the conventional way.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.